Polymerase chain reaction

The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to

amplify a single or a few copies of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA sequence.

Developed in 1983 by Kary Mullis PCR is now a common and often indispensable technique
used in medical and biological research labs for a variety of applications. These include DNA
cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of
hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and
paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was
awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.

The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the
reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA
fragments) containing sequences complementary to the target region along with a DNA
polymerase (after which the method is named) are key components to enable selective and
repeated amplification. As PCR progresses, the DNA generated is itself used as a template for
replication, setting in motion a chain reaction in which the DNA template is exponentially
amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an
enzyme originally isolated from the bacterium Thermus aquaticus). This DNA polymerase
enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by
using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers),
which are required for initiation of DNA synthesis. The vast majority of PCR methods use
thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of
temperature steps.

In the first step, the two strands of the DNA double helix are physically separated at a high
temperature in a process called DNA melting. In the second step, the temperature is lowered and
the two DNA strands become templates for DNA polymerase to selectively amplify the target
DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA
region targeted for amplification under specific thermal cycling conditions.

PCR principles and procedure:

PCR is used to amplify a specific region of a DNA strand (the DNA target). Most PCR methods
typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kb), although some
techniques allow for amplification of fragments up to 40 kb in size. The amount of amplified
product is determined by the available substrates in the reaction, which become limiting as the
reaction progresses.
A basic PCR set up requires several components and reagents. These components include:

 DNA template that contains the DNA region (target) to be amplified.

 Two primers that are complementary to the 3' (three prime) ends of each of the sense and
anti-sense strand of the DNA target.
 Taq polymerase or another DNA polymerase with a temperature optimum at around 70 °C.
 Deoxynucleoside triphosphates (dNTPs, sometimes called "deoxynucleotide
triphosphates"; nucleotides containing triphosphate groups), the building-blocks from which
the DNA polymerase synthesizes a new DNA strand.
 Buffer solution, providing a suitable chemical environment for optimum activity and stability
of the DNA polymerase.
 Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be
utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the
error rate during DNA synthesis[9]
 Monovalent cation potassium ions.
The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes
(0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes
to achieve the temperatures required at each step of the reaction (see below). Many modern
thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the
block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes
permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal
cyclers have heated lids to prevent condensation at the top of the reaction tube. Older
thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of
wax inside the tube.
Procedure
Figure 2: Schematic drawing of the PCR cycle. (1) Denaturing at 94–96 °C. (2) Annealing at
~65 °C (3) Elongation at 72 °C. Four cycles are shown here. The blue lines represent the DNA
template to which primers (red arrows) anneal that are extended by the DNA polymerase (light
green circles), to give shorter DNA products (green lines), which themselves are used as
templates as PCR progresses.
Typically, PCR consists of a series of 20-40 repeated temperature changes, called cycles, with
each cycle commonly consisting of 2-3 discrete temperature steps, usually three (Fig. 2). The
cycling is often preceded by a single temperature step (called hold) at a high temperature
(>90°C), and followed by one hold at the end for final product extension or brief storage. The
temperatures used and the length of time they are applied in each cycle depend on a variety of
parameters. These include the enzyme used for DNA synthesis, the concentration of divalent
ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.

 Initialization step: This step consists of heating the reaction to a temperature of 94–96 °C (or
98 °C if extremely thermostable polymerases are used), which is held for 1–9 minutes. It is
only required for DNA polymerases that require heat activation by hot-start PCR.
 Denaturation step: This step is the first regular cycling event and consists of heating the
reaction to 94–98 °C for 20–30 seconds. It causes DNA melting of the DNA template by
disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA
molecules.
 Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing
annealing of the primers to the single-stranded DNA template. Typically the annealing
temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-
DNA hydrogen bonds are only formed when the primer sequence very closely matches the
template sequence. The polymerase binds to the primer-template hybrid and begins DNA
formation.
 Extension/elongation step: The temperature at this step depends on the DNA polymerase
used; Taq polymerase has its optimumactivity temperature at 75–80 °C, and commonly a
temperature of 72 °C is used with this enzyme. At this step the DNA polymerase synthesizes
a new DNA strand complementary to the DNA template strand by adding dNTPs that are
complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the
dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The
extension time depends both on the DNA polymerase used and on the length of the DNA
fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA
polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if
there are no limitations due to limiting substrates or reagents, at each extension step, the
amount of DNA target is doubled, leading to exponential (geometric) amplification of the
specific DNA fragment.
 Final elongation: This single step is occasionally performed at a temperature of 70–74 °C for
5–15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is
fully extended.
 Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term
storage of the reaction.
Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers
were used to amplify a target sequence from three different tissue samples. No amplification is
present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the
target sequence. The gel also shows a positive control, and a DNA ladder containing DNA
fragments of defined length for sizing the bands in the experimental PCRs.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to
as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the
PCR products. The size(s) of PCR products is determined by comparison with aDNA ladder (a
molecular weight marker), which contains DNA fragments of known size, run on the gel
alongside the PCR products.
Medical applications
PCR has been applied to a large number of medical procedures:

 The first application of PCR was for genetic testing, where a sample of DNA is analyzed for
the presence of genetic disease mutations. Prospective parents can be tested for being genetic
carriers, or their children might be tested for actually being affected by a disease. DNA
samples for Prenatal testing can be obtained by amniocentesis, chorionic villus sampling, or
even by the analysis of rare fetal cells circulating in the mother's bloodstream. PCR analysis
is also essential to Preimplantation genetic diagnosis, where individual cells of a developing
embryo are tested for mutations.

 PCR can also be used as part of a sensitive test for tissue typing, vital to organ
transplantation. As of 2008, there is even a proposal to replace the traditional antibody-based
tests for blood type with PCR-based tests.[2]
 Many forms of cancer involve alterations to oncogenes. By using PCR-based tests to study
these mutations, therapy regimens can sometimes be individually customized to a patient.
Infectious disease applications
Characterization and detection of infectious disease organisms have been revolutionized by PCR:

 The Human Immunodeficiency Virus (or HIV), of antibodies to the virus circulating in the
bloodstream. However, antibodies don't appear until many weeks after infection, maternal
antibodies mask the infection of a newborn, and therapeutic agents to fight the infection don't
affect the antibodies. PCR tests have been developed that can detect as little as one viral
genome among the DNA of over 50,000 host cells. [3] Infections can be detected earlier,
donated blood can be screened directly for the virus, newborns can be immediately tested for
infection, and the effects of antiviral treatments can be quantified.

 Some disease organisms, such as that for Tuberculosis, are difficult to sample from patients
and slow to be grown in the laboratory. PCR-based tests have allowed detection of small
numbers of disease organisms (both live or dead), in convenient samples. Detailed genetic
analysis can also be used to detect antibiotic resistance, allowing immediate and effective
therapy. The effects of therapy can also be immediately evaluated.

 The spread of a disease organism through populations of domestic or wild animals can be
monitored by PCR testing. In many cases, the appearance of new virulent sub-types can be
detected and monitored. The sub-types of an organism that were responsible for earlier
epidemics can also be determined by PCR analysis.
Forensic applications
The development of PCR-based genetic (or DNA) fingerprinting protocols has seen widespread
application in forensics:

 In its most discriminating form, Genetic fingerprinting can uniquely discriminate any one
person from the entire population of the world. Minute samples of DNA can be isolated from
a crime scene, and compared to that from suspects, or from a DNA database of earlier
evidence or convicts. Simpler versions of these tests are often used to rapidly rule out
suspects during a criminal investigation. Evidence from decades-old crimes can be tested,
confirming or exonerating the people originally convicted.

 Less discriminating forms of DNA fingerprinting can help in Parental testing, where an
individual is matched with their close relatives. DNA from unidentified human remains can
be tested, and compared with that from possible parents, siblings, or children. Similar testing
can be used to confirm the biological parents of an adopted (or kidnapped) child. The actual
biological father of a newborn can also be confirmed (or ruled out).
Research applications
PCR has been applied to many areas of research in molecular genetics:

 PCR allows rapid production of short pieces of DNA, even when nothing more than the
sequence of the two primers is known. This ability of PCR augments many methods, such as
generating hybridization probes for Southern or northern blot hybridization. PCR supplies
these techniques with large amounts of pure DNA, sometimes as a single strand, enabling
analysis even from very small amounts of starting material.

 The task of DNA sequencing can also be assisted by PCR. Known segments of DNA can
easily be produced from a patient with a genetic disease mutation. Modifications to the
amplification technique can extract segments from a completely unknown genome, or can
generate just a single strand of an area of interest.

 PCR has numerous applications to the more traditional process of DNA cloning. It can
extract segments for insertion into a vector from a larger genome, which may be only
available in small quantities. Using a single set of 'vector primers', it can also analyze or
extract fragments that have already been inserted into vectors. Some alterations to the PCR
protocol can generate mutations (general or site-directed) of an inserted fragment.

 Sequence-tagged sites is a process where PCR is used as an indicator that a particular

segment of a genome is present in a particular clone. The Human Genome Project found this
application vital to mapping the cosmid clones they were sequencing, and to coordinating the
results from different laboratories.

 An exciting application of PCR is the phylogenic analysis of DNA from ancient sources,
such as that found in the recovered bones of Neanderthals, or from frozen tissues
of Mammoths. In some cases the highly degraded DNA from these sources might be
reassembled during the early stages of amplification.

 A common application of PCR is the study of patterns of gene expression. Tissues (or even
individual cells) can be analyzed at different stages to see which genes have become active,
or which have been switched off. This application can also use quantitative PCR to
quantitate the actual levels of expression